Over the past granting period we have for the first time obtained crystal structures of holoenzyme complexes of PKA. These complexes of the catalytic subunit bound to RI1, RII1 and RII have not only allowed us to understand how the catalytic subunit is inhibited by regulatory subunits but also how the cooperative activation of the holoenzyme is achieved by the binding of cAMP. In addition, we have begun to appreciate the molecular features that distinguish the PKA isoforms (Type I and II) and what the physiological consequences of those differences mean. During this period we also have delved into the molecular features that distinguish the AGC kinase subfamily from other protein kinases and have recognized, in particular, the unique features of the C-terminal tail and how it functions as a cis regulatory element for the kinase core. Finally, we have developed informatic tools to elucidate the conserved hydrophobic motifs that allow the kinase to function as a highly allosteric protein that is regulated by phosphorylation of its Activation Loop. In all of these cases, the catalytic subunit continues to serve as a prototype for the entire protein kinase superfamily. Over the next granting period we want to further understand the structure and dynamic properties of the catalytic subunit. In addition, we want to understand how the catalytic subunit is activated and how it interacts with other proteins and with membranes. The goal of Specific Aim I is to elucidate the structure and dynamic properties of the unphosphorylated C-subunit. Specific Aim II is to understand how the catalytic subunit is recognized and phosphorylated on its Activation Loop by an activating kinase, PDK1. In Specific Aim III we address the role of the N-terminal tail and especially the isoform-specific myristylation switch for mediating interactions with the kinase core, with other proteins, and with membranes. In Specific Aim IV we shall define how PKA recognizes substrates and inhibitors that dock in novel ways to the active site cleft. For each of these specific aims we shall utilize a variety of techniques. Peptide arrays will be used to help elucidate specific binding motifs and to define the boundaries and specificity of each motif. The functional importance of each motif will be validated in part by mutagenesis coupled with pull down assays, while Surface Plasmon Resonance will be used to quantify interactions. Hydrogen/deuterium exchange coupled with mass spectrometry will be used to identify interaction surfaces and allosteric pathways while small angle Xray scattering will be used to define global conformations and changes in global conformation. As always a major goal will be to validate our hypothesis by solving crystal structures of various complexes and activation states of the catalytic subunit. We believe that this will be an important new phase in our understanding of the complexity and allostery of the protein kinase superfamily and that our studies with PKA will continue to elucidate mechanisms that are relevant for all protein kinases. Although the details will be unique to each kinase, the underlying mechanisms are likely to be conserved. PUBLIC HEALTH RELEVANCE: Protein kinases account for nearly 2% of the human genome and are key switches for the regulation of the biology of all cells. They are also associated with many diseases and are one of the major drug targets today in biotech and pharmaceutical companies. PKA, the best characterized member of the protein kinase superfamily, serves as a prototype, and our structure/function studies of PKA continue to provide a foundation for understanding the structural and mechanistic properties of this entire enzyme family.